A new hot spot-based design strategy using bioisostere replacement is reported to rationally design nonpeptidic small-molecule inhibitors for protein-protein interactions. This method is applied to design new potent inhibitors for β-catenin/T-cell factor (Tcf) interactions. Three hot spot regions of Tcf for binding to β-catenin were quantitatively evaluated; the key binding elements around K435 and K508 of β-catenin were derived; a bioisostere library was used to generate new fragments that can match the proposed critical binding elements. The most potent inhibitor, with a molecular weight of 230, has a Kd of 0.531 μM for binding to β-catenin and a Ki of 3.14 μM to completely disrupt β-catenin/Tcf interactions. The binding mode of the designed inhibitors was validated by the site-directed mutagenesis and structure-activity relationship (SAR) studies. This study provides a new approach to design new small-molecule inhibitors that bind to β-catenin and effectively disrupt β-catenin/Tcf interactions specific for canonical Wnt signaling.